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EP0294996B1 - Système de détection fluorescent à balayage - Google Patents

Système de détection fluorescent à balayage Download PDF

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Publication number
EP0294996B1
EP0294996B1 EP88304979A EP88304979A EP0294996B1 EP 0294996 B1 EP0294996 B1 EP 0294996B1 EP 88304979 A EP88304979 A EP 88304979A EP 88304979 A EP88304979 A EP 88304979A EP 0294996 B1 EP0294996 B1 EP 0294996B1
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Prior art keywords
species
radiant energy
wavelength
set forth
filter
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German (de)
English (en)
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EP0294996A2 (fr
EP0294996A3 (fr
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Charles William Robertson, Jr.
Rudy Johan Dam
James Merrill Prober
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EIDP Inc
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EI Du Pont de Nemours and Co
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J1/00—Photometry, e.g. photographic exposure meter
    • G01J1/02—Details
    • G01J1/04—Optical or mechanical part supplementary adjustable parts
    • G01J1/06—Restricting the angle of incident light
    • G01J2001/062—Restricting the angle of incident light by fibre-optic packed bundle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J2003/1226—Interference filters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N2021/6417—Spectrofluorimetric devices
    • G01N2021/6421—Measuring at two or more wavelengths
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Definitions

  • This invention relates to a scanning fluorescent detection system and, more particularly, to apparatus suitable for use with a fluorescence-based DNA sequencer.
  • This system is capable of distinguishing among similar fluorophores with relatively low levels of emission.
  • a unique arrangement of a filter and fiber optic faceplate enables the system to monitor signals from relatively large detection areas containing multiple sample regions while still retaining the required optical characteristics of the combined filters.
  • DNA sequencing i.e., determining the sequence or order of the nucleotides or bases comprising the DNA, is one of the cornerstone analytical techniques of modern molecular biology.
  • the development of reliable methods for sequencing has led to great advances in the understanding of the organization of genetic information and has made possible the manipulation of genetic material, i.e., genetic engineering.
  • base sequence information which generally cannot be directly determined by physical methods must be converted into chain-length information which can be determined. This determination can be accomplished through electrophoretic separation. Under denaturing conditions (high temperature, urea present, etc.) short DNA fragments migrate through the electrophoresis medium as stiff rods. If a gel is employed for the electrophoresis, the DNA fragments will be sorted by size and result in a DNA sequence determination with single-base resolution up to several hundred bases.
  • the Sanger and Maxam-Gilbert methods for DNA sequencing are conceptually elegant and efficacious but they are operationally difficult, time-consuming, and often inaccurate. Many of the problems stem from the fact that the single radioisotopic reporter cannot distinguish between bases.
  • the use of a single reporter to analyze the sequence of four bases lends considerable complexity to the overall process.
  • the four sets of fragments produced by either Maxam-Gilbert or Sanger methodology are subjected to electrophoresis in four parallel lanes. This results in the fragments being spacially resolved along the length of the gel according to their size.
  • the pattern of labeled fragments is typically read by autoradiography which shows a continuum of bands distributed between four lanes often referred to as a sequencing ladder.
  • the ladder is read by visually observing the film and determining the lane in which the next band occurs for each step on the ladder.
  • Thermally induced distortions in base mobility in the gel can lead to difficulties in comparing the four lanes. These distortions often limit the number of bases that can be read on a single gel.
  • DNA fragments labeled with one or more fluorescent tags and excited with an appropriate light source give characteristic emissions from the tags which identify the fragments.
  • a fluorescent tag as opposed to a radioisotopic label allows one to specify a DNA fragment detection system that responds to the optical parameters characterizing tag fluorescence.
  • the use of four different fluorescent tags distinguishable on the basis of some emission characteristic (e.g., spectral distribution, life-time, polarization), allows one to uniquely link a given tag with the sequencing fragments associated with a given base. Once such a linkage is established, one can then combine and resolve the fragments from a single sample and make the base assignment directly on the basis of the chosen emission characteristic.
  • electrophoresis is chosen as a separation means, for example, a single sample containing DNA fragments with base-specific fluorescent tags can be separated in a single gel lane.
  • the fluorescence (emission) maxima are spread over a large wavelength range (approximately 100 nm) to facilitate discrimination among the four dyes, but, the absorption (excitation) maxima for the dyes are comparably spread. This makes it difficult to efficiently excite all four dyes with a single monochromatic source and adequately detect the resulting emissions.
  • a typical DNA sequencing experiment involves running multiple samples simultaneously in parallel lanes of a slab gel. Therefore, an excitation/detection system must also be able to monitor each lane of such a gel at substantially the same time.
  • a system must be capable of monitoring a detection zone which spans the majority of the usable gel width.
  • Typical sequencing gels have lanes that are 4-5 mm wide with 1-2 mm spacing between lanes. Therefore, in order to monitor a 10 lane gel, a detection system must excite and detect emissions from a region typically as wide as 70 mm.
  • the system described by Smith et al operates a fixed light beam and fixed detectors which together can monitor only a single point within the monitoring region.
  • the light beam In order to monitor more than one spatial position (lane or lane position) of a gel, either the light beam must be scanned while providing a means to detect the emissions from the dyes, or the gel must be physically shifted while holding the beam fixed.
  • moving the gel is not always practical since a large electrophoresis gel along with its associated buffer reservoirs are physically cumbersome.
  • moving the beam while the gel is stationary has its own problems since the detectors must remain closely coupled to sources of emission to prevent the entry of stray light and maximize collection of the emitted light.
  • One method of accomplishing this task is to physically move either of the two previously discussed detection systems and their associated optics and light beam so that several lanes in the gel are effectively scanned.
  • This type of system has the disadvantage of being mechanically complex while introducing additional noise into the system. Reliability and the high costs associated with this type of system would also be a concern.
  • the detectors must be either larger in size, or located further from the sample thus diminishing the collection efficiency.
  • the detection of closely spaced emission spectra of relatively low light intensities in the presence of a much more intense excitation source requires the selective transmission properties offered by interference filters.
  • both large detectors and large filters must be used.
  • large interference filters that collect light even a large solid angle are subject to transmission properties which vary with the angle of incidence of the light.
  • light impinging on the filter with an angle of incidence greater than about 22 degrees can experience significantly less rejection of the excitation light than light at normal incidence. Consequently, if the filter subtends a relatively large solid angle with respect to the source of emission, the overall excitation wavelength rejection properties of the filter will be compromised due to leakage of excitation light entering at the higher angles of incidence.
  • This invention finds use in a system for detecting the presence of radiant energy from different species, typically dye-labeled DNA, following separation in time and/or space, and identifying the species, the system having first detection means responsive to the radiant energy emitted by the species for generating a first signal that varies in amplitude in a first sense as a function of the nature of the species, second detection means responsive to the radiant energy for generating a second signal that varies in amplitude in a second sense different than the first sense as a function of the nature of the species, and third means responsive to the first and second signals for obtaining a third signal corresponding to the ratio of functions of the first and second signals, the amplitude of the third signal being indicative of the identity of each of the species.
  • the invention is an improvement of such system wherein the first and second means each include: a detector having a large solid entrance angle positioned adjacent to the species to receive radiant energy emitted from the species, and a wavelength selective filter means positioned between each respective detector and the species, each wavelength filter means having transmission vs. wavelength characteristics that are complementary, and wherein one of the first and second detection means includes a transmission filter means for rejecting radiant energy incident on a detector at an angle greater than a predetermined value.
  • the species are excited by a beam of radiant energy from a laser and the system includes means to separate molecules (typically fragments of DNA) labeled with emitting species of materials.
  • the detection means are positioned on opposite sides of the region propagating the laser beam of energy in which the beam is swept across the separation means to excite the species in sequence.
  • the wavelength selective filters have a transition in their transmission vs. wavelength characteristics centred at about the middle of the species' radiant energy spectra.
  • the transmission filter has an extra mural absorber among plural optical fibres positioned to have parallel generatrices transverse to the first and second detectors.
  • This system is optically efficient and does not require the use of lenses or other collection optics. It is capable of viewing, and, by the use of detectors having a wide entrance angle does view, large areas capable of accommodating plural electrophoresis lanes. These plural lanes are sequentially and repetitively scanned. Because of these efficiencies, the system can operate using low levels of emitted radiant energy. The only moving part required in the system is an optical element which effects the laser scanning. The use of the transmission filters and associated extra mural absorbers substantially reduce extraneous light impinging on the detector. The system is capable of detecting and distinguishing the radiant energy emitted from plural sources that emit energy at different but closely spaced wavelengths.
  • US-A-4580059 discloses a system for detecting the presence of different species and includes first and second detecting means generating respective first and second signals, means (in the form of a divider) responsive to the first and second signals to produce a third signal which corresponds to the ratio of the functions of the first and second signals, and interference filters in the detectors.
  • first and second detecting means generating respective first and second signals
  • means in the form of a divider
  • the detection of different species in the system of US-A-4580059 depends upon the extent to which the species quench the emission of a fluorescent indicator whose emission is then detected by the detector.
  • the radiation from very closely spaced emission bands may be detected using the system of this invention.
  • These closely spaced emissions are produced from preselected species which typically act as reporters and are irreversibly bound to materials that are to be analyzed.
  • Acceptable reporters are generally one or more species chosen for their ability to emit radiation over a narrow range of wavelengths, typically between a 50 and 100 nm range, preferably over a 20 to 50 nm range. Preferably, the peak maxima should be spaced no closer than 2 nm.
  • One reporter species may be capable of emitting energy at more than one wavelength, depending upon the manner of attachment to the materials of interest and the conditions of analysis in the system. However, individual reporters with unique emission characteristics in the system are more conventionally chosen to emit radiation in the wavelength range to be detected.
  • the invention may be used to detect any light emitting labelled samples and is particularly advantageous where the emission radiation has closely spaced wavelengths.
  • the invention may be used to detect, for example, fluorescence, chemiluminescence, and the like.
  • dye labelled DNA sequencing fragments are passed through an electrophoresis apparatus for separation.
  • the electrophoresis may be carried out by a suitable slab 10 arrangement typically having a thickness of about 0.3 mm and about 40 centimeters long and 15 centimeters wide. Other sizes may be used as appropriate.
  • This slab 10 has a suitable gel 11, typically 6% polyacrylamide; sandwiched between glass or low fluorescing plastic supports 12.
  • the slab 10 is typically placed in an upright position in a holder with the upper end of the slab 10 extending through and into an upper container 16 holding a buffer 24 and downwardly into a second container 14 also holding a buffer 18.
  • the buffer solution could be any suitable buffer such as that obtained from a solution consisting of .lM tris. .lM boric acid, and .05M Na2 EDTA, with a final pH of approximately 8.3. In this manner, the buffer contacts the gel at either end of the slab in order to make electrical contact therewith.
  • a sample containing reporter dye-labeled DNA fragments can be pipetted into cavities 15 that are created at the top of the gel 11 and define separation lanes.
  • the reservoir containers 14 and 16 are filled with buffer solutions 18 and 24.
  • An electrical circuit is then completed through the terminals 20 in reservoir containers 14 and 16.
  • a suitable electrical field typically 50 volts/cm
  • the positive electrode is located at the lover end of the slab to cause the DNA fragments to migrate downwardly. Under these conditions, as the fragments migrate through the gel they are separated spatially into bands (not shown).
  • Each of the four dyes prepared have reactive carboxy groups provided by a sarcosinyl moiety covalently bound to the 9-position of the parent compound, which are capable of forming covalent attachments with amine groups in linking moieties that join the dyes with selected nucleotides.
  • linking moieties found are a group of alkynylamine derivatives which contain a terminal amino group that can form covalent attachments with the dye carboxy groups.
  • a preferred linker is a 3-aminopropynyl derivative which is covalently attached to the 5-position of uracil (T) or cytosine (C), or to the 7-position of deazaguanine (d-G) or deazaadenine (d-A).
  • linker-nucleotide derivatives for use in the system of this invention were prepared with 2′, 3′-deoxyribonucleotides, which are known to serve as DNA chain terminating substrates for DNA polymerases. It was found that covalent attachment of the aminopropynyl-2′,3′-dideoxynucleotides to the fluorescent dyes in appropriate combinations, did not substantially diminish the chain terminating properties of the unsubstituted 2′,3′-dideoxynucleotides.
  • the four dye-linker-dideoxynucleotides A,G,C,T selected are illustrated by the structures: They were found to serve as useful chain terminating substrates for reverse transcriptase (avian myeloblastosis virus) in a modification of the well-known Sanger DNA sequencing method.
  • the classical Sanger method uses a primer, DNA template, DNA polymerase I (Klenow fragment), three unlabelled deoxynucleotides and one radiolabeled deoxynucleotide in each of four reaction vessels that each contain one of four 2′, 3′-dideoxynucleotides, which correspond to the four DNA bases (A,C,T,G).
  • Appropriate reaction conditions are created which allow the polymerase to copy the template by adding nucleotides to the 3′ end of the primer.
  • a multitude of reactions occur simultaneously on many primer copies to produce DNA fragments of varying length which all contain the radiolabel at appropriate nucleotides in each fragment, and which also irreversibly terminate in one of the four dideoynucleotides.
  • This set of fragments is typically separated on a polyacrylamide slab electrophoresis gel in four lanes, one lane corresponding to each of the four dideoxynucleotide reaction mixtures.
  • a photographic film is placed on the gel, exposed under appropriate conditions, and a DNA sequence is inferred from reading the pattern of bands created by the radiolabel on the film in order of their appearance in the four lanes from the bottom of the gel.
  • the modifications permitted by using these dye-labelled terminators include omitting the radiolabeled nucleotide and substituting the dye-labelled chain terminators for the unlabeled 2′,3′-dideoxynucleotides.
  • Reaction mixtures (actually a single reaction member can be used) will now contain fragments which are labeled on their 3′ ends with a fluorophore that corresponds to each of four DNA bases.
  • the reaction mixture(s) are combined and electrophoretically separated. Sequence is inferred by the order of appearance of bands being resolved in time or space that are revealed by the presence of fluorescent radiation.
  • the fluorescent dye-labelled dideoxynucleotides previously described are the preferred sources of closely spaced emitted radiation to be detected in the system and method of this invention.
  • An alternative source of emitted radiation which can also be useful in the system and method of this invention are the fluorophores described in the Smith et al. application. Their use would require selection of the appropriate laser frequencies and wavelengths of the preselected filters.
  • the optical arrangement for irradiating the lanes of the electrophoresis slab 10 is shown in Figure 2.
  • the system and apparatus of Figure 2 may be used with any fluorescent or other type system to distinguish between and measure the intensity of closely spaced emission radiation bands. However, it will be described by way of example of detecting the emissions from DNA fragments labeled with the particular reporters (dyes) set forth in the Prober et al. application, which application is incorporated herein by reference.
  • the dyes described in Prober et al. have peak emission wavelengths of about 505, 512, 519, and 526 nm. It includes a laser 30 which is selected to provide an exciting beam of radiation 32, with a specific wavelength determined as a function of the excitation wavelengths of the fluorophores used.
  • the specific source used with the dye fluorophores disclosed in Prober et al. is an argon ion laser with a wavelength of 488 nm and a 0.8 mm diameter light beam 32 operated at about 50 mW.
  • the light beam 32 passes through an excitation filter 34 and is then directed into scanning optics 36.
  • the filter 34 is selected to block out any undesired excitation wavelengths that could otherwise interfere with the detection process. However, for sufficiently spectrally pure lasers this filter may be omitted.
  • the scanning optics 36 include a prism or mirror 38 mounted on a fixed support (not shown), an astigmatic focusing lens 40, a second prism or mirror 43, and a cylindrical optic support 44 all mounted to the shaft of a stepping motor 46.
  • the beam 32, upon entering the scanning optics 36 is first directed downward by the prism 38 into the cylindrical opening of the optical support 44 and through the focusing lens 40.
  • Prism 38 serves to direct the beam from the laser into the scanning optics 36 thus facilitating convenient placement of the laser 30.
  • the light beam, passing through the focusing lens 40 is concentrated into an elliptical spot, in a preferred case of about 0.2 mm x 1-2 mm in cross-section.
  • the focused light beam 32 is directed through an exit aperture 42 by the second prism 43.
  • the optic support 44 is mounted to the shaft of the stepping motor 46 such that by actuating the stepping motor 46, the lens 40 and the prism 43 are rotated to cause the light beam 32 to angularly scan, in a horizontal plane perpendicular to the shaft axis and to the plane of the gel 10. This light beam 32 is directed at the detection zone 19 of the electrophoresis slab 10.
  • the light beam 32 upon entering the slab 10 excites the reporter material, here fluorescent dye labelled DNA fragments, as they migrate through the detection zone 19, causing them to fluoresce at wavelengths shifted from the excitation wavelength.
  • the peak emission wavelengths for the dyes described in Prober et al. are about 505, 512, 519, and 526 nm; however the system is adaptable to discriminate wavelengths associated with other sets of dyes with closely spaced emission bands.
  • a laser source is preferred since it allows a minimum of extraneous filtering and optics, other sources including a non-coherent source such as a xenon arc lamp could be used.
  • a reflective surface 50 (Fig. 3) can be positioned opposite from the excitation source.
  • a mirrored surface is deposited directly onto the outside of the outer plate 12 which supports or contains the gel.
  • the excitation light 32 which is not absorbed by the emitting species continues through the plate 12 and is reflected back towards the species by surface 50 to provide essentially twice the amount of excitation light.
  • the light given off by the fluorescent fragments is emitted in all directions so that light directed towards the reflective surface 50 is reflected also.
  • the net increase in fluorescent signal available for detection is approximately 4 times the amount available without the reflective surface.
  • the preferred method of providing a reflective surface is accomplished by coating the outside of the plate which supports the gel 10.
  • a mirror could be external to the glass but the increased number of interfaces that the light passes through would cause additional undesirable scattered excitation light.
  • the radiant energy or light emitted by the fluorescent species is collected by two suitably positioned upper and lower photodetector modules 52 and 54, respectively. These detector modules can be seen most clearly in Fig. 3 in which the details of their construction is shown.
  • the modules 52 and 54 are positioned above and below the plane of scanning of the light beam.
  • the modules are light tight in such a way as to eliminate stray light not directly coming from the excitation region.
  • Each module comprises a photomultiplier tube (PMT) 56 of conventional type having a wide entrance area.
  • a suitable photomultiplier tube is the Hamamatsu R1612.
  • Each module 52, 54 also has a separate wavelength selective filter 58 positioned between its PMT 56 and the fluorescent species in the gel slab 10.
  • the filters 58 preferably are custom interference filters which may be obtained from Barr Associates in Westford, MA, which have complementary transmission band characteristics as shown in Fig. 4 and are positioned to be transverse (preferably perpendicular on average) to the light 60 emitted from the species.
  • One filter 58 having a transmission characteristic denoted by the waveform A of Fig. 4 is seen to largely pass the lower emission wavelengths and reject the higher emission wavelengths.
  • the other filter 58 having a transmission characteristic denoted by the waveform B (Fig. 4) does precisely the reverse--it largely passes the higher wavelengths and largely rejects the lower wavelengths.
  • each module 52, 54 has a transmission filter 62 positioned between the wavelength selective filter 58 for that module and the emitting species. These filters can be reversed. Each transmission filter 62 rejects incident light entering the filter at off axis angles greater than a predetermined angle.
  • the two wavelength selective filters 58 enable the system to distinguish between closely spaced emission spectra. Light impinging on these wavelength filters 58 will either be transmitted, absorbed, or reflected.
  • the emission spectra of the four illustrative dyes selected, namely G505, A512, C519, and T526, are illustrated in Fig. 4.
  • the transmission filters 58 have been chosen to have transmissive characteristics (corresponding to curves A and B in Fig. 4 as described) which are complementary with that of the wavelength filter associated with the other photodetector in the wavelength region of the radiant energy emitted from the different species.
  • the two filters 58 are seen to have roughly complementary transmission vs. wavelength characteristics in the emission wavelength region of the four dyes, with the transition wavelengths occurring near the centre or middle of the species radiant energy spectra. As the fluorescence spectrum shifts from the shorter to longer wavelengths, the ratio of light transmitted through the upper filter 58 (in the drawing) to light transmitted through lower filter 58 (in the drawing) changes in a continuous manner.
  • the most suitable filters for this application are interference filters which have both a high relative transmission and high blocking at the excitation wavelength. Although these particular filters have been chosen to accommodate the particular dyes selected for this application, a different set of dyes could be suitably differentiated with other filter sets based on these principles.
  • the detectors and corresponding filters are selected to have a relatively large area for this application.
  • the detectors 56 preferably photomultiplier tubes, have large entrance windows nominally about 8 by 3.5 cm. In this way, a relatively large area 19 on the gel slab, can be monitored without the need for imaging optics which inherently create inefficiencies in the light collection and are sources of scattered light.
  • the detectors 56 in this system are positioned approximately 2-3 cm. from the emitting species such that multiple samples can be continuously monitored with high light collection efficiency. Under these conditions, it may be said that the detectors have a large solid entrance angle.
  • this problem is solved by the use of special transmission filters 62 coupled with the wavelength filters 58 such that the light impinging on the wavelength filters 58 is limited to a fixed range of angles close to the normal direction. The majority of the light which is impinging at an angle greater than a cut off value is either rejected or absorbed. If additional off-angled rejection is necessary, this invention can be combined with appropriate baffling, the use of colored filter glass or other known means.
  • a known device 62, useful for the transmission filters 62, having characteristics capable of rejecting off-angle light, is manufactured by INCOM located in Southbridge, Mass.
  • This device consists of a tightly packed bundle of optic fibers fused together, each fiber having a diameter of approximately 10 microns. The bundle is cut transversely across the fibers to produce an optic element of a desired thickness. Since each one of the fibers in the element has a nominal numerical aperture of about 0.35 only light impinging within a ⁇ 21° angle is allowed to be transmitted. Light impinging at angles greater than the acceptance angle is either rejected by first surface reflection or is passed through the cladding of the transmission fiber.
  • EMA extra mural absorber
  • light transmitted through the respective filter systems 62-58 is directed to the respective detectors 56.
  • the electrical signals from the detectors are then passed via respective preamplifiers 66,68 and to respective analog to digital (A/D) converters 70,72 and thence to a system controller 80.
  • the task of the system controller 80 may be performed by a small computer such as an IBM PC.
  • a function of the system controller 80, which is described by the flow diagram Figs. 4A, 4B and 4C is to compute the ratio of the two signal functions (the emission intensities on the PMT's 56 for each different emission wavelength) among other control tasks.
  • the wavelength filters 62 modulate the intensity of the signals in each of the different wavelength regions according to wavelength, i.e., from one detector, the shorter wavelength emissions will have a lower amplitude signal value and the longer wavelength emissions will have a higher amplitude. The reverse is true for the other detector since the filters have complementary characteristics as described.
  • a dye-labeled DNA fragment following separation in space in the gel slab, its emission spectra will generate two signals, one at the output of each detector 56, that vary in amplitude as a function of the emitted wavelength and time (because of the movement through gel slab 10).
  • the amplitude modulated light signals are at this point converted to electrical signals which are then digitized for such processing as described.
  • the functions of the two digital signals are ratioed, i.e., to obtain the quotient of the first and second signals of fluorescent light for each fluorophore.
  • the magnitude of the ratio signal is indicative of the identity of the species.
  • the ratio signals amplitudes for each dye tends to fall into distinct clusters or groupings which are readily distinguishable.
  • the term "closely spaced" in terms of the emission characteristics of the dyes or fluorophores is a somewhat relative term.
  • the minimum spacing between the center of emission of the dyes is large enough such that the difference in ratios of signals from the two detectors for any two adjacent dyes is distinguishable above the inherent system noise.
  • the filter characteristics can be further optimized. This can be accomplished by choosing filters with characteristics that change substantially over the different dye emission spectra. However, to distinguish a closely spaced group of dyes, it is preferable to have relatively sharp filter transitions that occur near the center wavelength of the group of dye emissions in order to evenly distribute the change in ratio of signals in the two filters for the different emission spectra.
  • the characteristics of an individual filter can also be fine-tuned to a degree by slightly varying the angle of incidence of the fiber optic faceplate output flux relative to the interference filter normal due to the idiosyncracies of interference filters as discussed previously.
  • the extra mural adsorption of the transmission filter 62 need be used for the wavelength filter having a passband closest to the excitation wavelength of the laser needs to be more than the other transmission filter.
  • the wavelength filter 58 having the passband A (Fig. 4) requires a transmission filter 62 with twice the EMA as used with the wavelength filter 58 having the passband B (Fig. 4). In some cases only one transmission filtere is needed.
  • the system controller 80 converts the digital signals received from the A/D converters 70 and 72 into DNA sequence information. In most cases, this will be done by a computer executing programs in real time. This means that data is processed and sequence information is determined concurrently with the acquisition of raw data from the detectors.
  • the operation of the system controller may be broken down into three interacting processes: data acquisition or input, data analysis, and output.
  • the processes interact by sharing data and by sharing timing information which keeps them "in step” and prevents them from interfering with one another.
  • the details of how these interactions are accomplished depend on the language and hardware chosen and is not of fundamental concern.
  • a general data acquisition process for a single channel is shown by the flow chart 5A.
  • the index i which points to the current acquired data, is initialized.
  • the program accepts an input which determines how long the run will take, i.e. the total number of data points I (total) .
  • the process enters an acquisition loop. Data are read from the detectors, digitized, and placed in the arrays as D1(i) and D2(i) for detector 52 and detector 54, respectively, acquired at time t(i). (For the purposes of this discussion, the two readings are simultaneous.)
  • the index i is incremented and compared to I (total) .
  • the acquisition loop is repeated. If i equals I (total) , the run is stopped.
  • the program could sense when to end the run automatically by measuring several performance parameters (such as signal/noise ratio, peak resolution, or uncertainty in assigning bases) at each peak of the run. If a combination of such factors failed to meet preset criteria, the run would be terminated by the computer.
  • the primary data input is the raw data from the detectors and the output is stored in the data arrays D1(i) and D2(i) which are shared between the acquisition and the data analysis processes.
  • This scheme is depicted schematically in Figure 5B.
  • the two programs run independently and simultaneously, some control information must be passed between them in order to maintain proper timing. For example, the processing steps cannot be allowed to overtake the acquisition step because it would then be attempting to process nonexistent data.
  • FIG. 5B and 5C The data processing algorithm depicted in Figures 5B and 5C is an example of a general scheme to detect and identify dye-labeled species. It is not meant to be all-inclusive. Rather, it illustrates the primary features that are necessary in developing any real analyzer program and is exemplary of applicants' preferred embodiment.
  • the program After initializing the processing index k (as distinct from the acquisition index i), the program enters a simple loop which reads data D1(k) and D2(k) from the data arrays provided by the acquisition process. The program then asks whether the current point is on a peak.
  • peak is meant in a general sense. A peak in D1 will generally be accompanied by a peak in D2. However, depending on the identity of the dye, the peaks in these two channels may differ considerably in intensity. They will, however, coincide in time. Therefore, a weighted average of the two signals, the stronger of the two signals, or some other combination of D1(k) and D2(k) could be used to define a "peak" in time.
  • the index k is incremented and compared with the acquisition index i. If k equals I (total) , the run is over and the program stops. If k is less than i, the next data points are fetched from the arrays D1 and D2 and the loop executes again. If k is equal to i, it means that processing has caught up with the data acquisition. In this event, the processing program waits a small period of time (typically a second) and again tests the values of k and i until processing can resume.
  • index m is incremented. Index m counts the number of points across the current peak.
  • the values D1(k) and D2(k) are placed in temporary array called D1peak(m) and D2peak(m), respectively.
  • the program then tests whether the current point is the last point of the peak. If this is not the last point on the peak, program control returns to the upper loop which increments k, tests its value against i, and reads the next pair of data from the arrays D1 and D2.
  • the peak counter is incremented and the program proceeds to determine the identity of the peak.
  • the result is the identity of the next base in the DNA sequence.
  • the program calculates the function W for the current peak as described above, using the arrays D1peak(m) and D2peak(m) as input data. Each nucleotide base will have associated with it a pair of peaks which give a characteristic W. Thus, based on the value of W for this peak, the program gives as output the DNA base identity A, T, C, G.
  • the peak point index m and the arrays D1peak and D2peak are reset to 0, and the program again enters the upper data acquisition loop as shown in Figure 6B.
  • the above scheme may be extended to a multi-sample scanner.
  • the laser beam would be moved to the next sample position before re-entering the data acquisition loop in Fig. 6A.
  • separate data arrays for example D1A(i), D1B(i)...D1Ki) and D2A(i), D2B(i)...D2K(i) for lanes A, B,...K

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Claims (15)

  1. Un système pour détecter la présence d'énergie radiante émise par différents émetteurs, après séparation dans le temps et/ou l'espace, comprenant:
       des premier moyens de détection (52) sensibles à l'énergie radiante émise par les émetteurs pour engendrer un premier signal qui varie en amplitude dans une première direction en fonction de la nature des émetteurs;
       des seconds moyens de détection (54) sensibles à l'énergie radiante pour engendrer un second signal qui varie en amplitude dans une seconde direction différente de la première en fonction de la nature des émetteurs; et
       des troisièmes moyens (80) sensibles au premier et ou second signaux pour obtenir un troisième signal correspondant au rapport des fonctions des premiers et seconds signaux;
       un filtre de sélection de longueur d'onde (58) étant disposé sur le trajet de l'énergie radiante dans chacun des premiers et seconds moyens de détection;
       caractérisé en ce que les différents émetteurs sont des espèces différentes dont on doit détecter la présence et l'identité, de telle façon que l'amplitude du troisième signal soit indicative de l'identité de chacune des espèces;
       en ce que les premiers et seconds moyens de détection comportent chacun:
       un détecteur (56) présentant un angle d'entrée solide important et positionné adjacent aux espèces devant recevoir l'énergie radiante émise des espèces pour engendrer l'un parmi le premier et le second signaux,
       en ce que le filtre de sélection de longueur d'onde (58), positionné sur le trajet de l'énergie radiante entre chaque détecteur correspondant (56) et les espèces, présente une caractéristique transmission/ longueur d'ondes qui est complémentaire de celle du filtre de sélection de longueur d'ondes associé à l'autre photodétecteur dans la zone des longueurs d'ondes de l'énergie radiante émises par ladite espèce différente; et
       en ce que l'un (52) des premier et second moyens de détection (52, 54) comporte un filtre de transmission (62) pour rejeter l'énergie radiante incidente sur le filtre de transmission sous un angle supérieur à une valeur prédéterminée.
  2. Un système selon la revendication 1 qui comporte également un laser (30) apte à diriger un faisceau d'excitation (32) d'énergie radiante vers les espèces, le faisceau laser d'énergie radiante présentant une longueur d'ondes située à l'intérieur de la zone d'excitation des espèces.
  3. Un système selon la revendication 2, dans lequel le filtre de transmission (6) est associé au filtre de sélection de longueur d'onde (58) présentant une bande passante qui est la plus proche de la longueur d'onde du faisceau laser (32) d'énergie radiante.
  4. Un système selon la revendication 2 et la revendication 3, dans lequel le premier et le second moyens de détection (52, 54) sont positionnés sur des faces opposées du faisceau de balayage laser.
  5. Un système selon l'une quelconque des revendications 1 à 4 qui comporte des moyens (15) aptes à séparer des fragments de DNA ou d'autres molécules marquées par des matériaux fluorescents, disposés pour être excités par l'énergie radiante.
  6. Un système selon la revendication 5 en combinaison avec la revendication 2, dans lequel l'énergie radiante qui excite les moyens aptes à séparer des fragments de DNA ou d'autres molécules marqués par des espèces de matériaux émettrices, est constitué par l'énergie radiante en provenance du laser.
  7. Un système selon l'une quelconques des revendications 1 à 6, caractérisé par l'absence d'une lentille entre les premier et second moyens de détection (52, 54) et les espèces émettrices.
  8. Un système selon l'une quelconque des revendications 1 à 7 qui comporte des moyens (36) pour étaler le faisceau laser (32) d'énergie radiante à travers les moyens de séparation (15) afin d'exciter les espèces en séquence.
  9. Appareil pour détecter la présence de rayonnement fluorescent dérivé de fragments de DNA ou d'autres molécules marquées en correspondance avec le type avec différentes espèces fluorescentes de matériaux et comprenant:
       des moyens de séparation (15) aptes à séparer spatialement les molécules;
       des moyens comprenant un laser (30) pour étaler dans un premier plan un faisceau d'énergie de rayonnement à travers les moyens de séparation pour exciter les espèces;
       des premier et second moyens de détection (52, 54), comportant:
    (i) des premiers et second photodétecteurs (56) ayant chacun un angle d'entrée solide important, disposés sur des faces opposées du premier plan pour convertir le rayonnement fluorescent émis par les espèces en premier et second signaux;
    (ii) interposés entre les photodétecteurs correspondants (56) et les moyens de séparation (15), des premier et second filtres de longueur d'onde (58) présentant des caractéristiques transmission/longueur d'onde qui sont complémentaires avec celles du filtre de longueur d'onde associé à l'autre photodétecteur dans la gamme de longueur d'onde de l'énergie radiante émise par lesdites espèces différentes; et
    (iii) un filtre de transmission (62) interposé entre l'un des photodétecteurs correspondant (56) et ses moyens de séparation, afin de rejeter le rayonnement incident sur le photodétecteur (56) sous un angle supérieur à une valeur prédéterminée; et
       des moyens (80) sensibles au premier et second signaux pour dériver un troisième signal correspondant au rapport des fonctions des premiers et second signaux, le troisième signal étant indicatif de l'identité de l'espèce fluorescente.
  10. Appareil selon la revendication 9, caractérisé par l'absence d'une lentille entre les premier et second photodétecteurs (56) et l'espèce émettrice.
  11. Un système selon l'une quelconque des revendications 1 à 8 ou un appareil selon la revendication 9 ou 10, dans lequel les filtres de sélection de longueur d'onde (58) présentent une caractéristique transmission/longueur d'onde centrée environ au milieu du spectre d'énergie radiante des l'espèces.
  12. Appareil selon la revendication 11, dans lequel la caractéristique de transmission est centrée approximativement au milieu du rayonnement fluorescent des espèces.
  13. Un système ou un appareil selon l'une quelconque des revendications 1 à 12, dans lequel le filtre de transmission (62) comporte un absorbeur d'ondes extérieur parmi une pluralité de fibres optiques disposées pour présenter des génératrices parallèles transversales aux premier et second moyens de détection.
  14. Un système ou un appareil selon l'une quelconque des revendications 1 à 13, dans lequel la valeur prédéterminée de l'angle de rejet du filtre de transmission (62) est d'environ 22°.
  15. Un système selon l'une quelconque des revendications 1 à 14, dans lequel les deux dits photodétecteurs (56) comportent ledit filtre de transmission (62) qui leur est associé.
EP88304979A 1987-06-12 1988-06-01 Système de détection fluorescent à balayage Expired - Lifetime EP0294996B1 (fr)

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US07/060,874 US4833332A (en) 1987-06-12 1987-06-12 Scanning fluorescent detection system
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Families Citing this family (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5571388A (en) * 1984-03-29 1996-11-05 Li-Cor, Inc. Sequencing near infrared and infrared fluorescense labeled DNA for detecting using laser diodes and suitable labels thereof
US5207880A (en) * 1984-03-29 1993-05-04 The Board Of Regents Of The University Of Nebraska DNA sequencing
US6086737A (en) * 1984-03-29 2000-07-11 Li-Cor, Inc. Sequencing near infrared and infrared fluorescence labeled DNA for detecting using laser diodes and suitable labels therefor
US5360523A (en) * 1984-03-29 1994-11-01 Li-Cor, Inc. DNA sequencing
US6207421B1 (en) 1984-03-29 2001-03-27 Li-Cor, Inc. DNA sequencing and DNA terminators
US6004446A (en) * 1984-03-29 1999-12-21 Li-Cor, Inc. DNA Sequencing
US5863403A (en) * 1984-03-29 1999-01-26 The Board Of Regents Of The University Of Nebraska Digital DNA typing
US4729947A (en) * 1984-03-29 1988-03-08 The Board Of Regents Of The University Of Nebraska DNA sequencing
US5135717A (en) * 1986-12-24 1992-08-04 British Technology Group Usa Inc. Tetrabenztriazaporphyrin reagents and kits containing the same
US5346670A (en) * 1986-12-24 1994-09-13 British Technology Group U.S.A. Inc. Phthalocyanine and tetrabenztriazaporphyrin reagents
US4919512A (en) * 1988-10-17 1990-04-24 Mcdonnell Douglas Corporation Wavelength shift switch and sensing system
US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
JPH083481B2 (ja) * 1989-06-07 1996-01-17 日立ソフトウェアエンジニアリング株式会社 蛍光式電気泳動パターン読み取り装置
FR2649487B1 (fr) * 1989-06-21 1991-09-20 Europhor Sa Capillaire d'electrophorese a dispositif optique incorpore
US5108179A (en) * 1989-08-09 1992-04-28 Myers Stephen A System and method for determining changes in fluorescence of stained nucleic acid in electrophoretically separated bands
IT1234466B (it) * 1989-09-20 1992-05-18 Consiglio Nazionale Ricerche Lidar a fluorescenza differenziale e relativo metodo di rilevamento
US5091652A (en) * 1990-01-12 1992-02-25 The Regents Of The University Of California Laser excited confocal microscope fluorescence scanner and method
US5274240A (en) * 1990-01-12 1993-12-28 The Regents Of The University Of California Capillary array confocal fluorescence scanner and method
JP2524243B2 (ja) * 1990-04-12 1996-08-14 日立ソフトウェアエンジニアリング株式会社 蛍光式電気泳動パタ―ン読み取り装置
JP2814408B2 (ja) * 1990-05-22 1998-10-22 日立ソフトウェアエンジニアリング 株式会社 蛍光パターン読み取り装置および蛍光パターン読み取り方法
US5119316A (en) * 1990-06-29 1992-06-02 E. I. Du Pont De Nemours And Company Method for determining dna sequences
JP2814409B2 (ja) * 1990-11-30 1998-10-22 日立ソフトウェアエンジニアリング 株式会社 多色泳動パターン読み取り装置
US5162654A (en) * 1991-02-01 1992-11-10 Wisconsin Alumni Research Foundation Detection apparatus for electrophoretic gels
WO1993019205A1 (fr) * 1992-03-19 1993-09-30 The Regents Of The University Of California Procede de marquage a plusieurs etiquettes permettant d'effectuer le sequencage de l'adn
US5294799A (en) * 1993-02-01 1994-03-15 Aslund Nils R D Apparatus for quantitative imaging of multiple fluorophores
US5424841A (en) * 1993-05-28 1995-06-13 Molecular Dynamics Apparatus for measuring spatial distribution of fluorescence on a substrate
US6035914A (en) * 1993-10-22 2000-03-14 Martin Marietta Energy Systems Inc. Counterfeit-resistant materials and a method and apparatus for authenticating materials
AU1562895A (en) * 1994-01-12 1995-08-01 Massachusetts Institute Of Technology Process for making xanthene or cubane based compounds, and protease inhibitors
JP3060001B2 (ja) * 1995-05-12 2000-07-04 日立ソフトウエアエンジニアリング株式会社 蛍光式電気泳動パターン読み取り装置
US5582705A (en) * 1995-05-19 1996-12-10 Iowa State University Research Foundation, Inc. Multiplexed capillary electrophoresis system
JP3012794B2 (ja) * 1995-10-02 2000-02-28 日立ソフトウエアエンジニアリング株式会社 スキャナ装置
US5699157A (en) * 1996-07-16 1997-12-16 Caliper Technologies Corp. Fourier detection of species migrating in a microchannel
US5871628A (en) * 1996-08-22 1999-02-16 The University Of Texas System Automatic sequencer/genotyper having extended spectral response
AU4920297A (en) 1996-10-25 1998-05-15 Applied Imaging, Inc. Multifluor-fluorescence in situ hybridization (m-fish) imaging techniques using multiple multiband filters with image registration
US5880473A (en) * 1997-07-28 1999-03-09 Applied Imaging, Inc. Multifluor-fluorescence in-situ hybridization (M-FISH) imaging techniques using multiple multiband filters with image registration
US6055451A (en) * 1997-12-12 2000-04-25 Spectrx, Inc. Apparatus and method for determining tissue characteristics
US20030135122A1 (en) * 1997-12-12 2003-07-17 Spectrx, Inc. Multi-modal optical tissue diagnostic system
JP2949220B2 (ja) * 1998-02-12 1999-09-13 工業技術院長 極微小顕微鏡分光装置
US6027628A (en) * 1998-02-23 2000-02-22 Yamamura; Hidetaka Gel cassette for electrophoresis
DE19816487A1 (de) * 1998-04-14 1999-10-21 Bodenseewerk Perkin Elmer Co Vorrichtung zum Nachweis eines Fluoreszenzfarbstoffs
US6078390A (en) * 1998-05-04 2000-06-20 General Scanning, Inc. Scanning system and method of operation for automatically setting detection sensitivity
CA2343401C (fr) * 1998-09-11 2009-01-27 Spectrx, Inc. Systeme optique multi-modal de diagnostic des tissus
US6036021A (en) * 1999-02-17 2000-03-14 C.C. Imex Package for electrophoresis gel
EP1169722A1 (fr) 1999-03-18 2002-01-09 Cambridge Research & Instrumentation, Inc. Systeme d'imagerie a sondes multiples a rendement eleve
US20040147843A1 (en) * 1999-11-05 2004-07-29 Shabbir Bambot System and method for determining tissue characteristics
US6696022B1 (en) * 1999-08-13 2004-02-24 U.S. Genomics, Inc. Methods and apparatuses for stretching polymers
US6462816B1 (en) * 2000-07-21 2002-10-08 Symyx Technologies, Inc. Parallel capillary electrophoresis system having signal averaging and noise cancellation
JP3690271B2 (ja) * 2000-11-29 2005-08-31 株式会社島津製作所 核酸の塩基配列決定のためのマトリックス値を得る方法
JP2002277326A (ja) * 2001-03-19 2002-09-25 Nireco Corp 分光測光装置
US6844563B2 (en) * 2001-05-22 2005-01-18 Matsushita Electric Industrial Co., Ltd. Fluorescence detecting device with integrated circuit and photodiode, and detection method
JP2004537053A (ja) * 2001-07-25 2004-12-09 アプレラ コーポレイション 電気泳動検出システムにおける時間遅延積分
US7262017B2 (en) 2001-09-14 2007-08-28 Torrey Pines Institute For Molecular Studies Diagnostic markers for ischemia
US7383134B2 (en) * 2002-01-15 2008-06-03 Piper James R Method and/or system for analyzing biological samples using a computer system
AU2003228277B2 (en) * 2002-03-05 2006-06-29 Caliper Life Sciences, Inc. Mixed mode microfluidic systems
US20050143933A1 (en) * 2002-04-23 2005-06-30 James Minor Analyzing and correcting biological assay data using a signal allocation model
US6999173B2 (en) * 2003-09-25 2006-02-14 Ffa Sciences Llc Method and apparatus for ratio fluorometry
US7601510B2 (en) * 2004-03-22 2009-10-13 Ffa Sciences Llc Development and use of fluorescent probes of unbound analytes
WO2006036388A2 (fr) 2004-08-23 2006-04-06 U.S. Genomics, Inc. Systemes et procedes de detection et d'analyse de polymeres
US20060257938A1 (en) * 2005-05-10 2006-11-16 Kleinfeld Alan M Method of screening for drugs that block ligand binding to a lipid binding protein
EP1886104A4 (fr) * 2005-05-20 2011-11-16 Koninkl Philips Electronics Nv Capteur de chromaticite multicolore
EP2044403A1 (fr) * 2006-07-18 2009-04-08 TIR Technology LP Procédé et appareil de détermination d'intensités et de longueurs d'onde maximales de lumière
US7381972B1 (en) * 2006-07-24 2008-06-03 Science Applications International Corporation System and method for light fluorescence detection
US9164109B2 (en) 2006-10-27 2015-10-20 Alan Kleinfeld Use of probes for unbound metabolites
US8999636B2 (en) * 2007-01-08 2015-04-07 Toxic Report Llc Reaction chamber
WO2009009127A2 (fr) * 2007-07-12 2009-01-15 U.S. Genomics, Inc. Procédé et système de transfert et/ou de concentration d'un échantillon
DE102008011013B4 (de) * 2008-02-25 2014-11-13 Mevitec Gmbh Verfahren und Einrichtung zur komplexen Stoffwechselanalyse
US8361716B2 (en) * 2008-10-03 2013-01-29 Pathogenetix, Inc. Focusing chamber
WO2010088514A1 (fr) 2009-01-30 2010-08-05 Micronics, Inc. Système portable de détection de fluorescence à gain élevé
CA2786569C (fr) 2010-01-29 2019-04-09 Micronics, Inc. Cartouche microfluidique «de l'echantillon au resultat»
DE102012002086A1 (de) 2012-02-06 2013-08-08 Carl Zeiss Meditec Ag Verfahren zum Untersuchen von biologischem Gewebe und Vorrichtungen zum Untersuchen und Behandeln des Gewebes
US8685708B2 (en) 2012-04-18 2014-04-01 Pathogenetix, Inc. Device for preparing a sample
US9028776B2 (en) 2012-04-18 2015-05-12 Toxic Report Llc Device for stretching a polymer in a fluid sample
WO2014100743A2 (fr) 2012-12-21 2014-06-26 Micronics, Inc. Films à faible élasticité pour utilisation micro-fluidique
CN104919191B (zh) 2012-12-21 2019-07-09 精密公司 流体回路及相关的制造方法
US20150346097A1 (en) 2012-12-21 2015-12-03 Micronics, Inc. Portable fluorescence detection system and microassay cartridge
EP2994750B1 (fr) 2013-05-07 2020-08-12 PerkinElmer Health Sciences, Inc. Dispositifs microfluidiques et méthodes de séparation du sérum et de compatibilité croisée du sang
CN105189750B (zh) 2013-05-07 2020-07-28 珀金埃尔默健康科学有限公司 使用粘土矿物和碱性溶液制备含核酸样品的方法
WO2014182847A1 (fr) 2013-05-07 2014-11-13 Micronics, Inc. Dispositif pour la préparation et l'analyse d'acides nucléiques

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3588257A (en) * 1968-10-14 1971-06-28 Us Navy Flame photometric method and apparatus for quantitatively analyzing material specimens
US3764512A (en) * 1972-05-02 1973-10-09 Singer Co Laser scanning electrophoresis instrument and system
US4152723A (en) * 1977-12-19 1979-05-01 Sperry Rand Corporation Method of inspecting circuit boards and apparatus therefor
US4162405A (en) * 1978-05-23 1979-07-24 Britton Chance Flying spot fluoro-meter for oxidized flavoprotein and reduced pyridine nucleotide
US4451149A (en) * 1980-07-28 1984-05-29 Noeller Hans Guenter Polarization fluoroimmunoassay apparatus
US4421860A (en) * 1980-10-07 1983-12-20 The Regents Of The University Of California Homogeneous fluoroimmunoassay involving autocorrelation processing of optically sensed signals
AT390840B (de) * 1983-06-08 1990-07-10 Avl Verbrennungskraft Messtech Verfahren zur fluorimetrischen bestimmung der konzentration von in einer substanz enthaltenen stoffen und anordnung zur durchfuehrung dieses verfahrens
JPS60197698A (ja) * 1983-12-20 1985-10-07 カリフオルニア・インステイテユ−ト・オブ・テクノロジ− アミノ誘導化されたオリゴヌクレオチドの合成
GB2155176B (en) * 1984-01-16 1988-05-11 California Inst Of Techn Sequencing dna
US4729947A (en) * 1984-03-29 1988-03-08 The Board Of Regents Of The University Of Nebraska DNA sequencing
US4651011A (en) * 1985-06-03 1987-03-17 At&T Technologies, Inc. Non-destructive method for determining the extent of cure of a polymer
CA1340806C (fr) * 1986-07-02 1999-11-02 James Merrill Prober Methode, systeme et reactifs pour le sequencage de l'adn

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DE3851910D1 (de) 1994-12-01
AU1762088A (en) 1988-12-15
DE3851910T2 (de) 1995-05-24
US4833332A (en) 1989-05-23
EP0294996A2 (fr) 1988-12-14
EP0294996A3 (fr) 1991-04-10
AU601048B2 (en) 1990-08-30
JPS63317770A (ja) 1988-12-26
IL86695A (en) 1992-01-15
CA1299772C (fr) 1992-04-28
IL86695A0 (en) 1988-11-30

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